We are interested in a fundamental issue in the development of multicellular organisms, i.e., how a few undifferentiated cells assume their identities and form distinct patterns. We have chosen to address this issue in the context of Arabidopsis flow development. In particular, we are studying how precursor cells assume their identifies and develop into stamens and carpels, the male and female reproductive organisms of the flower. For a long time, AG, a MADS domain transcription factor, was the only known protein involved in this developmental process. It is not clear how a single transcription factor leads to the eventual formation of complex organs such as stamens and carpels. It is our long-term goal to fully dissect the AG pathway in order to understand this complex developmental process. Two new members of the AG pathway, HUA1 and HUA2, were isolated from our enhancer screen using a weak ag allele. HUA2 was cloned with a map-base strategy. It codes for a putative transcription factor and transcriptional co-activator of AG. Significant progress has been made towards the cloning of HUA1. This proposal is aimed at uncovering the molecular mechanisms underlying the functions of HUA1 and HUA2 in Arabidopsis flower development. First, HUA2 will be studied at the protein level. HUA2 protein localization pattern within the plant will be determined. Experiments will be performed to study the physical interaction between AG and HUA2, which was observed in our preliminary studies. Our genes whose products interact with HUA2 will be isolated with a yeast two-hybrid screen. Second, HUA1 will be cloned and studied at the molecular level. Its RNA localization patterns within the plant will be determined. Potential interactions among HUA1, HUA2 and AG proteins will be investigated. Third, additional mutant alleles of HUA1 and HUA2 will be isolated, especially severe loss-of-function mutations, which will help solve some pending issues regarding the functions of HUA1 and HUA2 in the AG pathway. A combination of a molecular screen of T-DNA insertional lines and a Hua1 Hua2 enhancer screen will be employed for this purpose. A detailed understanding of a homeotic pathway in Arabidopsis will be an invaluable addition to our knowledge of developmental process in general. Plant and animal homeotic pathways can be compared to derived evolutionary principles. In addition, studying plant development in a simple organism like Arabidopsis will likely expedite our understanding of more complicated organisms such as humans. In light of the fact that HUA2 shares common motifs with genes in C. elegans, Drosophila, mice and humans, it will not be surprising that the molecular functions of some plant genes in homeotic pathways are conserved in animals.